Research in this laboratory is concerned with understanding the biology of mammalian DNA repair, and the mechanisms by which unrepaired damage is translated into biologic effects such as cell lethality, mutation, and carcinogenesis. Particular attention is given to the relationship between DNA repair and trinucleotide repeat instability, which is a hall mark feature in various severely degenerate diseases such as Huntington~s disease,, fragile X syndrome, Friedreich~s ataxia and myotonic dystrophy. The broad an long term objective of this proposal is to gain insight as to the biologic significance of the relationship between the structure- specific nucleases and genomic instability. In contrast to the significant progress in identifying triple repeats associated diseases, very little is known about the molecular mechanism which triggers trinucleotide repeat instablitly. Furthermore, the is very limited information about t the key molecules that are essential for the prevention of triple repeat expansion. Based upon yeast genetic studies and our biochemical results, we hypothesize that human flap- endonuclease (FEN-1), which is essential for DNA replication and repair, plays a pivotal role in the prevention of trinucleotide expansion in human. The specific aims of this study are: 1) to evaluate the FEN-1's ability to process trinucleotide repeats in vitro. 2) to establish the structure/function basis for FEN-1's ability to process trinucleotide repeats, and 3) to determine the role of FEN-1 in the prevention of trinucleotide instability in vivo. The experimental design to be used in the pursuit of these aims will employ; 1_ in vitro flap-endonuclease assay with various model substrates of the Okazaki fragment containing trinucleotide repeats, 2_ a comprehensive site-directed mutagenesis study to understand the mechanism of FEN-1's ability to recognize and process trinucleotide repeats, and 3) a novel glowingcytometry/green fluorescent protein- based trinucleotide repeat assay in vivo. The results of this study will provide a detailed mechanistic understanding of the trinucleotide repeat instablitlity in mammalian genome.